Communication between various leukocyte populations and the unique stroma of different organ microenvironments is important for understanding the regulation and optimization of development of innate immune responses and their relationship to metastasis formation and disease-induced inflammation. IL-12 and IL-18 are potent immunoregulatory cytokines for natural killer (NK), NKT, and T cells, and have very distinctive effects on the NKT/NK subsets in different organs. Administration of IL-12 results in an overall increase in total liver-associated leukocytes, a rapid reduction in the total number of detectable NKT cells in the liver, and a later reduction in NK cells. The ability of IL-12 to recruit T cells to the liver is dependent on MIP1alpha. Conversely, IL-18 increases the number of NKT and NK cells, and this increase in liver leukocytes is dependent on CXCR3 binding chemokines. Studies of the interactions between NK and NKT cells are difficult because the only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosyl ceramide (alpha-GalCer) which also has a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, ?GalCer (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies employing CD1d tetramers loaded with ?GalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared to alpha-GalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, ?GalCer (C12), in contrast to alpha-GalCer failed to increase NK cell size, number and cytolytic activity. Also in contrast to alpha-GalCer, ?GalCer (C12) is a poor inducer of IFN-gamma, TNFalpha GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alpha-GalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while ?GalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show ?GalCer (C12) can effectively discriminate between NKT and NK-mediated responses in vivo. Studies of these different TcR-binding ceramides are now in progress to better delineate the contributions of NK vs NKT cells to antimetastatic activity induced in the liver after hydrodynamic delivery of the IL-2 gene to mice.